Measurement of the photoreceptor pointing in the living chick eye

Abstract

The chick eye is used in the study of ocular growth and emmetropization; however optical aberrations in the lens and cornea limit the ability to visualize fine retinal structure in living eyes. These aberrations can be corrected using adaptive optics (AO) allowing for cellular level imaging in vivo. Here, this capability is extended to measure the angular tuning properties of individual photoreceptors. The left eyes from two White Leghorn chicks (Gallus gallus domesticus) labeled chick A and chick B, were imaged using an AO flood illuminated fundus camera. By translating the entrance pupil position, the same retinal location was illuminated with light of varying angles allowing for the measurement of individual photoreceptor pointing. At 30° nasal from the pecten tip, the pointing direction for both chicks was towards the pupil center with a narrow distribution. These particular chicks were found to have a temporal (T) and inferior (I) bias in the alignment with peak positions of (0.81 T, 0.23 I) and (0.57 T, 0.18 I) mm from the pupil center for chicks A and B respectively. The rho, ρ, values for the major, ρL, and minor, ρs, axes were 0.14 and 0.17mm(-2) for chick A and 0.09 and 0.20mm(-2) for chick B. The small disarray in the alignment of the chick photoreceptors implies that the photoreceptors are aligned to optimize the light entering the eye through the central portion of the pupil aperture. The ability to measure pointing properties of individual photoreceptors will have application in the study of eye growth and various retinal disorders.

Keywords: Adaptive optics; Chick; In vivo retinal imaging; Photoreceptors; Retina; Stiles–Crawford Effect of the First Kind.

Figure 1

The entrance pupil locations used during the AO imaging procedure are indicated by the red circles. The diameter of the entrance pupil at each location was 0.3 mm. By translating the pupil plane iris in the delivery arm, the same retinal location was imaged through the 25 entrance pupil locations. The labels indicate the pupil plane coordinates in mm.

Figure 2

The two dimensional Gaussian construction used by Gorrand and Delori (Gorrand & Delori, 1995). The dashed circle and lines represent the edge of chick’s dilated pupil and the horizontal and vertical axes in the pupil plane respectively. The solid lines and circles show the distribution of photoreceptors at the pupil plane. θ is the rotation angle of the x’y’ coordinates of the Gaussian function with respect to the xy coordinates of the subject’s pupil. (x0, y0) is the location of the peak in the pupil plane (i.e. the pointing direction of the photoreceptors).

Figure 3

Images of the same retinal location at 5 entrance pupil locations centered at the SCE-I peak for chick A. Image intensities have been equalized to show the photoreceptor detail. The same three photoreceptors indicated by the colored circles are shown for each image. Each image is 100 × 100 μm in size at the retina and is the registered sum of 10 images. The labels indicate the entrance pupil location in mm from pupil center.

Figure 4

Equivalent images for chick B. Each image is 100 × 100 μm in size at the retina. Each image is a registered sum of 10 images.

Figure 5

Retinal images for chick A at the 22 different pupil locations used in the analysis. The images have been plotted on the same intensity scale to illustrate the variation in brightness as the entrance pupil position was moved. As the entrance beam moved away from the central locations, the images became darker due to the decreased photoreceptor reflection to light rays incident at oblique axes. The inset figure (top left) shows the same information but scaled over a unity range, the SCE peak position (0.5, 0) being set to 1. Each image is 25 × 25 μm in size at the retina. Registered sum of 10 images for each pupil location. The axes denotes the entrance pupil position in mm.

Figure 6

The 20 retinal images for chick B taken through the 20 entrance pupil positions. The images have been plotted on the same intensity scale to illustrate the variation in brightness as the entrance pupil position was moved. Each image is 25 × 25 μm in size at the retina. Registered sum of 10 images for each pupil location.

Figure 7

Photoreceptor analysis procedure for chick A. (A) the registered image from position (0, 0) is analyzed using a semi-automated Matlab script to determine the photoreceptor centers, denoted by the red dots. (B) the pointing direction of each photoreceptor relative to the pupil center is determined using Eq. 1 with the red lines giving the direction and magnitude of the pointing. (C) shows the deviation in pointing from the ensemble average i.e. the disarray among the 288 photoreceptors analyzed. (D) shows the projected intercept for each individual photoreceptor (denoted by a red cross) at the pupil plane and (E) shows the overall Gaussian fit to average of the individual photoreceptor fitting parameters, the scaling is in arbitrary units. The retinal images are 100 × 100 μm in size at the retina. The white scale bar in (B) and (C) indicate a pupil plane deviation of 2mm. The results are summarized in Table 2.

Figure 8

Similar analysis procedure for chick B. (A) shows the pointing direction of each photoreceptor relative to the pupil center with the red lines showing the direction and magnitude of the pointing. (B) shows the deviation in pointing from the ensemble average i.e. the disarray among the 276 photoreceptors analyzed. (C) shows the projected intercept for each individual photoreceptor (denoted by a red cross) at the pupil plane and (D) shows the overall Gaussian fit to average of the individual photoreceptor fitting parameters, the scaling is in arbitrary units. The retinal images are 100 × 100 μm in size at the retina. The white scale bar in (A) and (B) indicate a pupil plane deviation of 2mm. The results are summarized in Table 2.